To understand the biochemical mechanisms of eukaryotic DNA replication, we have developed a unique soluble cell-free system which catalyzes SV40 DNA replication dependent on exogenously added SV40 form I DNA and SV40 large T-antigen. The system is characterized by de novo initiation of SV40 DNA replication in vitro. An in vitro complementation assay for SV40 large T-antigen based on this in vitro SV40 DNA replication showed that the extract prepared from SV40-infected CV-1 cells did contain a functional SV40 large T-antigen, whereas the extract prepared from SV80 cells (a SV40 transformed human cell line) did not contain a functional large T-antigen. Furthermore, the extracts prepared from the rapidly growing (log phase) CV-1 cells which were not infected with SV40, also supported SV40 DNA replication in vitro even in the absence of SV40 large T-antigen. Extracts prepared from uninfected stationary (Go phase) CV-1 cultures did not support SV40 DNA replication.